This invention relates to processes for producing glycoproteins in eukaryotic cell culture. The invention provides cell culture processes which preserve oligosaccharide structures of nascent glycoproteins and greatly facilitate the recovery of glycoproteins containing oligosaccharides terminating in one or more sialic acid residues from the cell culture.
Many eukaryotic cell surface- and secreted proteins are post-translationally processed to incorporate N-linked and O-linked carbohydrate (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thought to subserve a variety of functions including augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion (Fieldler and Simons (1995) Cell 81:309-312; Helenius (1994) Mol. Biol. of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473; Caton et al., (1982) Cell 37:417-427; Alexander and Elder (1984) Science 226:1328-1330; Flack et al. (1994) J. Biol. Chem. 269:14015-14020). In higher organisms, the nature and extent of glycosylation can markedly affect the circulating half-life and bio-availability of secreted proteins by mechanisms involving receptor mediated uptake and clearance (Ashwell and Morrell (1974) Adv. Enzymol. 41:99-128; Ashwell and Harford (1982) Ann. Rev. Biochem. 51:531-54). Receptor systems have been identified that are thought to play a major role in the clearance of serum proteins through recognition of various sugar components of the oligosaccharide on the glycoproteins (Stockert (1995) Physiol. Rev. 75:591-609; Kery et al., (1992) Arch. Biochem. Biophys. 298:49-55). Since the terminal sialic acid component affects absorption, serum half life, and clearance from the serum as well as the physical, chemical and immunogenic properties of the glycoprotein (Parekh, R. B., suira; Varki, A., (1993) Glycobiology 3:97-100; Paulson, J. (1989), TIBS, 14:272-276; Goochee, et al., (1991) Biotechnology 9:1347-1355; Kobata, A, (1992) Eur. J. Biochem. 209:483-501) production strategies which preserve the terminal sialic acid component can advantageously lengthen protein bioavailability and serum half-life.
Much attention has been paid to factors which affect glycosylation during recombinant protein production such as growth mode (adherent or suspension), fetal bovine serum in media formulation, culture density, oxygenation, pH, ammonium concentration, purification schemes and the like (Werner, R. and Noe, W. (1993), Drug Res. 43:1134-1249; Hayter et al., (1992) Biotech. and Bioeng. 39:327-335; Borys et al., (1994) Biotech and Bioeng. 43:505-514; Borys et al., (1993) Bio/Technology 11:720-724; Hearing et al., (1989) J. Cell Biol. 108:339-353; Goochee et al., in Frontiers in Bioprocessing II, Todd et al., eds (1992) American Chemical Society pp.199-240; U.S. Pat. No. 5,096,816; Chotigeat, W., (1994) Cytotech. 15:217-221; Gawlitzek et al., (1998) Biotech. Bioeng. 57:518-528). Several groups have investigated the process parameters that surround the production of recombinant proteins and especially the effect of media composition in various production strategies (Park et al., (1992) Biotech. Bioeng. 40:686-696; Cox and McClure, (1983) In Vitro, 19:1-6; Mizutani et al., (1992) Biochem. Biophys. Res. Comm. 187:664-669; Le Gros et al. , (1985) Lymph. Res. 4(3):221-227). For example U.S. Pat. No. 5,705,364 discloses methods of altering the sialic acid content of a glycoprotein by controlling factors that affect cell productivity, such as the addition of an alkanoic acid to the culture medium, controlling osmolality of the culture medium, and controlling growth temperature.
Another means of affecting sialic acid content of glycoprotein is by controlling the activity of cellular sialidases (U.S. Pat. No. 5,510,261). Sialidases are cytosolic and membrane-associated enzymes that cleave sialic acid from glycosyl moiety of a glycoprotein. The activity of synaptosomal membrane-associated sialidase of bovine brain was inhibited by copper ion at high concentration following pre-saturation in situ (Yohe, H. C. and Rosenberg, A. (1978) Neurochemical Research (1978) 3:101-113). Cytosolic and membrane-associated sialidase I was shown to be inhibited by copper ion at approximately 1 mM concentration in partially purified extracts from rat liver or rat skeletal muscle (Miyagi, T. et al. (1993) Glycoconjugates Journal 10:45-49).
Culture media and additives frequently contain trace elements and metal ions such as copper for optimal cell growth (for example, xe2x80x9cProtease Peptone 2 and 3,xe2x80x9d xe2x80x9cPrimatone RLxe2x80x9d and xe2x80x9cPrimatone HSxe2x80x9d, which are commercially available (Sheffield, England; Difco, USA); Japanese Patent No. 93JP-0171420; International Publication No. WO 90/03430; for composition of various media, for example, DMEM and HAM F12 media, see culture media formulations in American Type Culture Collection Catalogue of Cell Lines and Hybridomas, Sixth Edition, 1988, pages 346-349). A serum-free medium containing Zn (5-100 xcexcM) or Cu (0.1-50 xcexcM) for growth of vascular endothelial cells was proposed in Japanese Patent No. 247618. Lanier and Volkman reported an increase in yield of baculovirus expression vectors from recombinant baculoviruses generated in insect (lepidopteran) tissue culture in a medium containing 2 mM CuSO4 (Lanier, L. M. and Volkman, L. M. (1996) In Vitro 32(3) Pt.2:8A). Hultberg et al. found that metals increased the amount of glutathion reduced in HeLa cell culture medium and that copper ion also increased the amount of reduced homocysteine in the medium at copper ion concentrations that did not interfere with cell growth (1-100 xcexcm). (Hultberg, B. et al. (1997) Toxicology 117:89-97). Divalent metal ions such as Cu2+ and Zn2+ in soluble form at a concentration of 70-120 mg/L have been added to serum and the serum added to cell culture medium to promote cell growth (German Patent No. 155 328).
However, eukaryotic cells, especially mammalian cells such as Chinese hamster ovary (CHO) cells have been shown to be sensitive to Cu2+ concentration in culture media (Camakaris et al., (1995) Human Molecular Genetics 4:2117-2123; Steinebach and Wolterbeek (1994) J. Inorganic Biochemistry, 53:27-48; German Patent 155 328; Sakai, Y. et al. (1994) Cytotechnology 14 (Suppl. 1):7-36). CHO-K1 cells were found to exhibit an LD50 of 126 xcexcM Cu2+ in copper-supplemented culture medium (Camakaris, J. et al. (1995) Human Molecular Genetics 4:2117-2123).
The present invention provides for processes for producing glycoproteins by mammalian cell culture which maintain the sialic acid component of oligosaccharides of glycoproteins produced.
The present invention provides processes for producing glycoproteins by eukaryotic cell culture which provide a glycoprotein product containing oligosaccharides terminating in one or more sialic residues. Advantageously, the cell culture processes of the present invention allow for the recovery of a glycoprotein product whose oligosaccharides are not compromised by degradative events associated with standard cell culture procedures. The processes of the present invention overcome the problem of desialylation of a glycoprotein""s oligosaccharide side chains that are associated with standard glycoprotein production methods. Advantageously the invention provides economic and commercial benefits through the recovery of greater useful quantities of glycoprotein product.
Accordingly, the invention provides processes for producing glycoproteins by eukaryotic and especially mammalian cell culture comprising culturing a host cell expressing a glycoprotein in the presence of copper ion in a cell culture medium in a concentration effective to minimize the loss of sialic acid. The present invention therefore provides various cell culture processes to preserve particular glycoforms of glycoproteins produced in mammalian cell culture.
The present invention provides, in a particular embodiment, for producing a glycoprotein in mammalian cell culture by adding an effective amount of Cu2+ to a culture medium in which cells producing the glycoprotein are grown. According to one aspect of the invention, the concentration of Cu2+ is between approximately 50 xcexcM and 5 mM. Preferably the Cu2+ concentration in the medium is between about 0.1 mM and about 2 mM, preferably at least approximately 250 xcexcM. More preferably the copper ion concentration is in the range of about 0.1 mM to about 1 mM, especially in the range of about 0.1 mM and about 0.5 mM and more preferably at least approximately 350 xcexcM, and at least approximately 380 xcexcM. The foregoing parameter is controlled to affect the mature glycoprotein sialic acid content.
In a particular aspect of the invention, the host cells producing the glycoprotein are grown in a serum-free media. Preferably the host cell is a mammalian host cell and preferably Chinese hamster ovary (CHO) cells including, but not limited to CHO K1, CHO pro3xe2x88x92, CHO DG44, CHO DUXB11 and CHO DP12 cells. Other mammalian host cells useful in the method of the invention include, but are not limited to, mouse myeloma cells, NS0, and hybridoma cells, such as mouse hybridoma cells baby, hamster kidney (BHK) cells, COS cells HeLa cells, C127 cells, mouse L cells, 293 cells and Ltkxe2x88x92 cells.
In a preferred embodiment, the invention provides for a process for increasing the sialic acid content of the mature glycoprotein recovered from a mammalian host cell culture comprising contacting the cells expressing the glycoprotein with Cu2+ in the culture medium at a concentration that inhibits or prevents desialylation in the cell culture medium. Preferably the Cu2+ concentration is at least 150 xcexcM, more preferably at least approximately 380 xcexcM. Preferably the host cells are CHO cells. According to this aspect of the present invention, culturing the host cell at a concentration of about 380 xcexcM provides for the recovery of a glycoprotein with an increased sialic acid content.
The invention further provides, in a particular embodiment, for a cell culture process with two or three phases of cell culture. The invention therefore provides a process for controlling the sialic acid content of a glycoprotein produced by mammalian host cell culture comprising the steps of culturing a host cell which expresses the glycoprotein in a growth phase for a period of time and under such conditions that cell growth is maximized. According to this aspect of the present invention, the growth phase is optionally followed by a transition phase in which cell culture parameters for the desired sialic acid content of the mature glycoprotein are selected and engaged. The growth phase or transition phase is followed by a production phase of the cell culture wherein production parameters optionally selected and engaged in the transition phase are maintained and glycoprotein product is produced and harvested. Adding Cu2+ to the cell culture medium after the growth phase, during the transition phase or advantageously at the start of the production phase following the transition phase produces a protein with increased amount of sialic acid which is maintained.